EP0527941B1 - Identification of novel drugs and reagents - Google Patents

Identification of novel drugs and reagents Download PDF

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Publication number
EP0527941B1
EP0527941B1 EP91910623A EP91910623A EP0527941B1 EP 0527941 B1 EP0527941 B1 EP 0527941B1 EP 91910623 A EP91910623 A EP 91910623A EP 91910623 A EP91910623 A EP 91910623A EP 0527941 B1 EP0527941 B1 EP 0527941B1
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EP
European Patent Office
Prior art keywords
cells
oligonucleotide
infectious agent
oligonucleotides
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
EP91910623A
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German (de)
English (en)
French (fr)
Other versions
EP0527941A4 (enrdf_load_stackoverflow
EP0527941A1 (en
Inventor
Christopher K. Mirabelli
Timothy Vickers
David J. Ecker
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Ionis Pharmaceuticals Inc
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Isis Pharmaceuticals Inc
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Publication date
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Publication of EP0527941A1 publication Critical patent/EP0527941A1/en
Publication of EP0527941A4 publication Critical patent/EP0527941A4/xx
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Publication of EP0527941B1 publication Critical patent/EP0527941B1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it

Definitions

  • This invention relates to the identification, preparation and use of novel therapeutic, diagnostic and research compositions and to methods for their use. More particularly, this invention relates to novel antisense compositions, compositions which can interact with nucleic acids by hybridization to effect their function and to cause changes in biological activity thereby. Methods for identifying such antisense compositions are provided which lead to novel therapeutic, diagnostic and research compositions and to methods for diagnosis, treatment and experimentation.
  • oligonucleotides or oligonucleotide analogues which are designed to bind in a specific fashion to--which are specifically hybridizable with-- a specific mRNA by hybridization.
  • Such analogues are intended to inhibit the activity of the selected mRNA-- to interfere with translation reactions by which proteins coded by the mRNA are produced- by any of a number of mechanisms.
  • the inhibition of the formation of the specific proteins which are coded for by the mRNA sequences interfered with have been hoped to lead to therapeutic benefits.
  • antisense oligonucleotides have been introduced into antisense oligonucleotides to increase their therapeutic activity. Such modifications are designed to increase cell penetration of the antisense oligonucleotides, to stabilize them from nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotide analogues in the body, and to improve their pharmacokinetic properties. At present, however, no generalized antisense oligonucleotide therapeutic or diagnostic scheme has been found. Prior efforts have been limited by the inability to deliver a sufficient quantity of antisense oligonucleotide into appropriate cells for effective activity to take place.
  • a further object is to identify oligonucleotides useful for the determination of the status of bodily functions of animals.
  • Yet another object is to identify oligonucleotides which are useful as research reagents such as for blocking gene expression of particular RNA molecules.
  • An additional object of the invention is to identify loci of nucleic acids which control the expression of polypeptides having a significant effect on a bodily function of an animal.
  • a further object is to identify and provide such oligonucleotides without the need for prior knowledge of the sequence of nucleic acid for which hybridization is desired.
  • antisense oligonucleotides are currently being investigated as potential therapeutic agents, it is not clear how to select the best possible nucleotide sequence to inhibit the expression of a particular mRNA. Moreover, in many cases it is even difficult to decide what gene to target for antisense inhibition to alter the course of a disease. The method of the present invention overcomes these difficulties.
  • methods for identifying oligonucleotides capable of hybridizing with nucleic acid of an infectious agent comprising providing a plurality of vectors which include substantially randomly sequenced oligonucleotides and incorporating those vectors into cells.
  • the cells are infected with the infectious agent and provided with conditions for growth of the cells.
  • Cells which are resistant to the infection are then identified.
  • the nucleic acid sequence of the included oligonucleotide is determined for those cells resistant to the infection.
  • the substantially randomly sequenced oligonucleotides are prepared through solid state synthesis or otherwise and comprise from about 10 to about 100 nucleic acid subunits.
  • the cells used are preferably selected to be easily infectable by the infectious agent.
  • reagents are prepared having as at least a component thereof the nucleic acid sequence of the inserted oligonucleotide which has been identified as coming from a cell resistant to the infectious agent.
  • D1 and D2 describe the screening of cDNA libraries to identify unknown genes by a "subtractive" hybridisation technique whereby antisense forms of the cDNA library made from cellular mRNA are reintroduced into the cell from which the library was made and phenotypic alterations determined.
  • Figure 1 depicts a schematic structure of the vector ISIS RG-1.
  • the present invention provides a method of identifying anti-sense oligonucleotide sequences that inhibit gene expression of an infectious or other agent.
  • Expression vectors containing oligonucleotide sequences formed by random synthesis are transformed into cells.
  • the cells are then infected with the infectious or other agent.
  • Oligonucleotides that inhibit gene expression of the infectious agent and which are thus useful in suppressing the effects of the agent are then selected by selecting cells that do not exhibit effects of infection with the infectious agent; for example, the cells do not die, the cells grow at an increased rate, or exhibit other behavior suggestive of modulation of the infection.
  • the sequence of the oligonucleotide having the ameliorative effect is identified. Identification may be accomplished by recovering the vector and sequencing the region containing the inserted nucleic acid material.
  • the cells, expression vectors and method of selecting for the oligonucleotide sequence will be determined by the type of infectious or other agent. For example, if it is desired to identify anti-sense oligonucleotides to a herpesvirus, the cell type chosen will be one that can be infected with the particular herpesvirus, and the expression vector will be one that is compatible with the cell chosen.
  • the term "random" as applied to sequences of nucleic acid comprehends truly random oligonucleotides formed through solid state synthesis. Even this protocol need not be completely and statistically random in fact, however. Thus, enrichment of oligonucleotides in certain bases may be desired in accordance with some embodiments of this invention.
  • the oligonucleotide fragments of portions are generally of a size as to be effective in the performance of this invention. Generally, from about 20 to 100 bases are employed for the synthetically generated random sequences.
  • Expression vectors suitable for use in the methods of the invention include commercially available plasmid vectors such as pMAMM-NEO (Clonetech) and other expression vectors. It is preferable that the plasmid expression vector contain at least one gene to allow construction and recovery of the vector, such as an antibiotic resistance gene such as the ampicillin resistance gene. It is also preferable that the plasmid expression vector contain an inducible promoter and transcriptional initiations region to express the random sequences and a polyadenylation signal to stabilize the random sequences. This may or may not include an intron for more efficient expression.
  • a preferred plasmid expression vector is ISIS RG-1 which is illustrated schematically in figure 1.
  • This vector contains the neomycin gene for G418 selection of stably transformed cells in culture and the ampicillin gene for bacterial amplification. Inserts can be directionally cloned by cutting at the Hind III and Xba I sites, releasing a stuffer fragment. The Hind III site is immediately 3' to the RSV promoter and the xba I site is immediately 5' of the bovine growth hormone (BGH) poly (A) site.
  • BGH bovine growth hormone
  • Random sequences can be prepared by use of a DNa synthesizer to generate a length of DNA with an equimolar mixture of A, G, C, T at each position.
  • the complimentary strand can be generated using a DNA primer and DNA polymerase.
  • the expression vectors are inserted into cells susceptible of infection with the infectious agent.
  • the cells may already be infected with the infectious agent or they may be infected with the infectious agent after insertion of the expression vector.
  • the expression vectors are inserted into the cells by standard methods, such as calcium phosphate transfection or electroporation.
  • neommyosin resistant cells can be propagated to confluency in 10 cm tissue culture dishes. When confluency is reached the entire population is infected with HSV-1 at a low MOI. Media is changed at frequent intervals following the infection to reduce the frequency of secondary infection. Cells which survive the infection are grown to confluency, then infected a second time in the same manner. This process is repeated until all cells survive an infection or until individual resistant colonies are selected.
  • the sequences of the oligonucleotides having activity are determined. Identification of the oligonucleotide sequence may be accomplished by several methods. The oligonucleotide may be removed from the expression vector and sequenced. Alternatively, the oligonucleotide sequence may be amplified in the cell by polymerase chain reaction, using appropriate primers, and the sequence of the amplified sequence determined.
  • the following method describes a scheme to generate random-synthetic oligomers of defined length, to screen for sequences to specifically inhibit HSV infection of mammalian cells in culture.
  • the vector used will be ISIS RG-1. This vector contains the neomycin gene for G418 selection of stably transformed cells in culture and the ampicillin gene for bacterial amplification. Inserts can be directionally cloned by cutting at the Hind III and xba I sites, releasing a stuffer fragment. The Hind III site is immediately 3' to the RSV promoter and the Xba I site is immediately 5' of the bovine growth hormone (BGH) poly (A) site. (see fig. 1)
  • a random population of oligonucleotides will be produced such that the 5' end of each oligomer consists of the Hind III site, d(CAAGCTTG). This is followed by approximately 25 nt of random sequence, then the tail sequence d(TCTAGAGAAAAA), creating an Xba I site and poly A tail.
  • a primer complementary to the 3' end sequence, 5'd(TTTTTCTCTAGA)3', will then be used as a primer for the synthesis of complementary strands to each of the random oligomers produced creating double strand molecules.
  • the population of oligomers will then be subject digestion with Hind III and xba I to give cohesive ends compatible with the vector.
  • the plasmids will be transfected as a population into CV-1 or HeLa cells using the method of Chen and Okayama. Stable transformants will be selected on the basis of resistance to G418. The population of stably transformed cells will then be infected with HSV. Surviving colonies presumably will be expressing mRNA which is interacting to specifically block HSV infection. The plasmid DNA responsible for the effect can then be isolated from the transformed cells by PCR using Hind III and xba I primers.

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EP91910623A 1990-05-01 1991-04-15 Identification of novel drugs and reagents Expired - Lifetime EP0527941B1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US51724090A 1990-05-01 1990-05-01
US517240 1990-05-01
PCT/US1991/002574 WO1991017266A1 (en) 1990-05-01 1991-04-15 Identification of novel drugs and reagents

Publications (3)

Publication Number Publication Date
EP0527941A1 EP0527941A1 (en) 1993-02-24
EP0527941A4 EP0527941A4 (enrdf_load_stackoverflow) 1994-04-27
EP0527941B1 true EP0527941B1 (en) 1996-09-18

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EP91910623A Expired - Lifetime EP0527941B1 (en) 1990-05-01 1991-04-15 Identification of novel drugs and reagents

Country Status (10)

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EP (1) EP0527941B1 (enrdf_load_stackoverflow)
JP (1) JP2706568B2 (enrdf_load_stackoverflow)
AT (1) ATE143056T1 (enrdf_load_stackoverflow)
AU (1) AU7974791A (enrdf_load_stackoverflow)
CA (1) CA2081984A1 (enrdf_load_stackoverflow)
DE (1) DE69122246T2 (enrdf_load_stackoverflow)
DK (1) DK0527941T3 (enrdf_load_stackoverflow)
ES (1) ES2092568T3 (enrdf_load_stackoverflow)
GR (1) GR3021418T3 (enrdf_load_stackoverflow)
WO (1) WO1991017266A1 (enrdf_load_stackoverflow)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5965376A (en) * 1991-10-31 1999-10-12 Matritech, Inc. Nuclear matrix protein fluid assay

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0558697A1 (en) * 1991-06-28 1993-09-08 Massachusetts Institute Of Technology Localized oligonucleotide therapy
DE69333377T2 (de) * 1992-06-22 2004-10-21 Matritech Inc Neuartige marker maligner zelltypen in der inneren nukleären matrix
US5919647A (en) * 1992-10-16 1999-07-06 Hamamatsu Photonics K.K. Methods and apparatuses for examining pathogen resistance of plant, for evaluating ability to impart pathogen resistance to plant, and for evaluating agricultural chemical
JP3231098B2 (ja) * 1992-10-16 2001-11-19 浜松ホトニクス株式会社 植物の病害抵抗性検定方法および装置、病害抵抗性付与能力の評価方法および装置、農薬の評価方法および装置
US6455247B1 (en) 1996-01-23 2002-09-24 Board Of Trustees Of The Leland Stanford Junior University Methods for screening for transdominant effector peptides and RNA molecules
US6365344B1 (en) 1996-01-23 2002-04-02 The Board Of Trustees Of The Leland Stanford Junior University Methods for screening for transdominant effector peptides and RNA molecules

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5310893A (en) 1986-03-31 1994-05-10 Hoffmann-La Roche Inc. Method for HLA DP typing

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5965376A (en) * 1991-10-31 1999-10-12 Matritech, Inc. Nuclear matrix protein fluid assay
US6162608A (en) * 1991-10-31 2000-12-19 Matritech, Inc. Nuclear matrix protein fluid assay
US6740494B2 (en) 1991-10-31 2004-05-25 Matritech, Inc. Nuclear matrix protein fluid assay

Also Published As

Publication number Publication date
JP2706568B2 (ja) 1998-01-28
DE69122246T2 (de) 1997-02-06
GR3021418T3 (en) 1997-01-31
CA2081984A1 (en) 1991-11-02
AU7974791A (en) 1991-11-27
EP0527941A4 (enrdf_load_stackoverflow) 1994-04-27
EP0527941A1 (en) 1993-02-24
JPH05505529A (ja) 1993-08-19
DE69122246D1 (de) 1996-10-24
DK0527941T3 (da) 1996-10-07
WO1991017266A1 (en) 1991-11-14
ATE143056T1 (de) 1996-10-15
ES2092568T3 (es) 1996-12-01

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